Aquaculture, i.e. the specific cultivation of aquatic animals in a closed environment, has received serious and widespread investigation within the past twenty years as a result of an increasing awareness of the world food shortage. During this period several groups have investigated the potential of various types of aquaculture in conjunction with various species of aquatic animals. Several types of aquaculture have been studied including "Open Environment Aquaculture", wherein the aquatic animals are enclosed in large cages in their natural habitat, and "Pond Culture", wherein large ponds are stocked with aquatic animals. In conjunction with these studies several aquatic species have been studied including finfish, most notably catfish and trout, and shellfish, most notably oysters, each of which species has been raised with some degree of success. More recently, however, investigators have come to recognize the significant potential offered by the aquaculture of crustaceans, specifically shrimp, in "Controlled Environment Aquaculture" (CEA), i.e. the intensive culture of dense animal populations in an artificial and highly controlled system.
One embodiment of a CEA system is described in U.S. Pat. No. 3,998,186 to Hodges, the disclosures of which are herein incorporated by reference. CEA systems such as the one described in the Hodges patent may include one or more elongated waterways, or raceways, which contain an appropriate aquatic environment in which shrimp may be cultured. In addition, such a system may be equipped with an aquatic medium exchange means whereby new aquatic medium may be exchanged for used medium present in the raceway. Furthermore, the CEA system may include either a feed introduction means or a source of natural feed, or both, and an areation device for maintaining a desired level of dissolved oxygen in the medium. Finally, a CEA system may include a clear, or partially opaque, canopy which covers the raceway and which may, if desired, allow sunlight to enter the aquatic medium.
The saltwater penaeid shrimp has been demonstrated to be culturable in captivity and to reach optimum market sizes in a few months. Hatchery techniques and reproductivity in captivity have been demonstrated for penaeid shrimp both in the United States and abroad. Furthermore, since the natural habitat of penaeid shrimp is salt water, the potential appears to exist for aquaculture of this species either in seawater or in brackish water from drilling sites. Penaeid shrimp, thus, appear to offer significant potential as a crop for Controlled Environment Aquaculture.
Freshwater shrimp appear to offer less promise as potential CEA species due to their lower fecundity, their aggressive behavior, and their intolerance to crowding. However, they do offer promise in less intensive aquaculture systems such as Pond Culture.
Before the Controlled Environment Aquaculture, or, indeed, any type of aquaculture, of shrimp can be considered to be commercially practical, however, most, if not all, of the procedures necessary for their culturing must be optimized in order that the venture will be economically viable. Thus an economical method for feeding the shrimp and an economical method for providing the optimum growth and survival rates within an aquatic environment must be developed. In this regard, one of the most important aspects of the aquaculture of shrimp is the treatment and prevention of diseases.
In high density populations, such as those utilized in CEA, diseases may be carried into the system by new aquatic medium which is circulated through the system by the aquatic medium exchange means and by wild females which are introduced into the system as spawners. In addition, certain disease-like conditions may result from nutritional deficiencies or abnormalities. Since the high population densities present in such systems make the animals more susceptible to stress (and thus to disease organisms present in aquatic medium), it is of the utmost importance to develop techniques for early diagnosis of such diseases and the most effective therapeutic agents for their treatment.
Several diseases and disease-like conditions have been found to afflict both wild and artificially cultured shrimp, with the severity of certain of these individual conditions being greater in artificial cultures due to the much higher population densities encountered in such systems. (see Disease Diagnosis and Control in North American Marine Aquaculture, Sinderman, C. J. Ed., Elsevier Scientific Publ. Co., New York, 1977, pp. 8-95.) Among the various diseases and disease-like conditions which afflict shrimp, gill disease presents an especially serious threat to their survival. (See Lightner, D. V., "Gill Disease: A Disease of Wild and Cultured Penaeid Shrimp", presented at the 66th International Council for the Exploration of the Sea, Copenhagen, Denmark, 1978.) The term gill disease encompasses a complex of several diseases which are developed by penaeid shrimp, both in wild environments that receive industrial or marine sewage, such as near-shore or estaurine waters, and in aquacultural systems that necessarily contain high levels of feed and/or natural waste due to the high population densities present. The majority of the organisms involved in gill disease of penaeid shrimp are free living organisms and are, thus, not true pathogens, but infestations. Nevertheless, when attached to, and abundant on, the gills, these organisms cause mortality indirectly by interfering with respiration either by preventing sufficient water-flow over the gills or by reducing gas exchange across gill surfaces.
Among the several organisms which are included under the general term of gill disease is a filamentous micro-organism which bears a close resemblance to Leucothrix mucor. This filamentous micro-organism has been identified on wild P. aztecus taken from estaurine waters near Galveston, Texas and on the gills and appendages of cultured P. stylirostris, P. californiensis, P. vannamei, and P. mondon. In every case the L. mucor-like micro-organism appears as unbranched thin tapering filaments of 3 to 5 .mu.m diameter at the base, and tapering to 1-3 .mu.m diameter apically. Filaments are attached to the shrimp's cuticle by an inconspicuous holdfast and extend to a length of from a few micrometers up to a millimeter or more. The filaments are sheathed by a thin inconspicuous sheath, and consist of many cells which are shorter in their axial measurement than they are in diameter. Gonidia are developed apically on the filaments. In gonidia formation, the apical region of a filament develops a beaded appearance caused by constriction of the outer cell wall at the transverse septa. Gonidia appear to be released by abcission from the filament either as single cells or as short chains, and to act as the transfer, or infective, stage of the micro-organism which ultimately locates and attaches to a new substrate.
This micro-organism, from penaeid shrimp does not appear to have been isolated and grown in pure culture, and, thus, its classification as L. mucor must be considered to be tentative. However, the L. mucor-like filaments found on penaeid shrimp appear to be morphologically indistinguishable from L. mucor as described by Harold and Stanier [Biological Reviews 19: 49-58 (1955)], Snellen and Raj [J. Bateriology, 101: 240-249 (1970)], and Skelton, et al. [J. Mar. Biol. Assoc. U.K., 55: 795-800 (1975)]. Because of its similarity to Leucothrix mucor, the filamentous micro-organism described above will be referred to as L. mucor. Nevertheless it is the micro-organism as described, rather than as tentatively identified as L. mucor, which has been found to be one of the major organisms in what is termed herein as gill disease.
Another filamentous organism is often present with L. mucor on the gills and appendages of penaeid shrimp. This organism is smaller in diameter (0.5 to 1.0 .mu.m) and typically shorter than L. mucor, and is composed of individual cells that are longer than they are wide. This organism has been isolated from the gills and grown in pure culture. From such observations, this organism has been tentatively identified as Cytophaga sp. However, as was the case with L. mucor, the exact taxonomic position of this organism has not been conclusively proven. Thus, although this organism will be referred to herein as Cytophaga sp. herein, it is the organism as described, rather than as tentatively identified, which has been found in close association with L. mucor in penaeid shrimp.
Other filamentous organisms have been observed on the gills, appendages and general body surface of cultured penaeids, albeit with less frequency than L. mucor or Cytophaga sp. Certain filamentous blue-green algae including Lyngbya sp., Oscillatoria sp., and Spirulina subsalsa have been occassionally observed in sufficient amounts on the gills of cultured shrimp to have caused mortality, presumably due to respiratory failure. These organisms, with the exception of Spirulina, closely resemble L. mucor in morphology but can be readily distinguished by their larger diameter (greater than 4 .mu.m) and the presence of chlorophyl pigments which give them a definite green to blue-green color.
Maintenance of good water quality and low population densities will minimize the presence of these filamentous forms of gill disease in penaeids. However, in high density aquaculture, such as CEA, chemotherapeutic treatments are often necessary to prevent shrimp mortalities resulting from these filamentous organisms. At present, the treatment of choice is a water soluble algaecide containing triethanolaminecopper(II) chelate manufactured under the trademark Cutrine-Plus by Applied Biochemists of Mequon, Wis. Cutrine-Plus has been used at 0.1-1.0 ppm copper(II) ion in 24 hr "flow through" treatments, (i.e. where aquatic media exchange is continued and the agent is constantly metered into the exchanging media) or at 0.25-0.5 ppm copper(II) ion in 4 to 6 hour "static" treatments (i.e. where aquatic media exchange is discontinued and the agent is added to the static environment and maintained in contact with the shrimp for the specified time after which aquatic media exchange is resumed). Permanganate ion (as potassium permanganate) at 5 to 10 ppm in 1 hr static treatments given every five to ten days has also been found to be effective to reduce these filamentous organisms. Thus, both Cutrine-Plus and permanganate ion are effective in treating gill disease, but, when used at the higher end of the stated dosage range, each treatment may cause shrimp mortality due to gill damage or agent toxicity.
The term gill disease also includes disease conditions caused by colonial peritrich protozoans, primarily Zoothamnium sp. and less commonly Epistylis sp. and Vorticella sp. These organisms have been reported to cause mortalities in cultured shrimp, are common in the epifauna of marine and brackish water environments, and are occassionally found on wild penaeids in nutrient rich estaurine waters.
Shrimp with heavy infestations of Zoothamnium sp., have a fuzzy-appearing mat on the surface of the gills, appendages, and occassionally on the carapace. Microscopic examination of wet mounts made from scrapings of these areas show Zoothamnium sp., to be branched colonial organism. Zoothamnium sp. have been observed on all four species of cultured penaeid shrimp discussed above.
Although less common among cultured shrimp, the protozoans Epistylis sp. and Vorticella sp. are occassionally observed on shrimp with gill disease. These organisms are similar in appearance to Zoothamnium sp., but Zoothamnium sp. may be distinguished from Epistylis sp. and Vorticella sp. because the former organism possesses a continuous myoneme that connects the stalks of each trophont within the colony so that the colony may contract as a unit. Epistylus lacks a contractile stalk, and, while Vorticella sp. is often colonial and possesses a contractile stalk, it does not have a continuous myoneme connecting individual members of the colony. Hence, the Vorticella sp. colonies do not contract as a unit as does Zoothamnium.
Like L. mucor, these peritrich protozoans cause disease and death in penaeid shrimp when they are abundant on the gills, although they cause no discernable histopathology and evoke no inflammatory response. As with the filamentous forms of gill disease, shrimp mortality due to protozoans, is presumed to result from hypoxia due to reduced respiratory efficiency of the gills.
Numerous other gill disease organisms are seen occassionally in association with L. mucor filaments on the gills of shrimp. Included among these organisms are various blue-green algae, eg. Enteromorpha sp. and Ulva sp., several diatom species, and numerous species of Gram negative bacteria, most commonly Vibrio sp. Various saprophytic fungi such as the imperfect fungus Fusarium solani, (which is responsible for a disease called black gill disease when it infects gill tissues) may also be present.
In order to ensure the economic viability of an aquaculture system such as the CEA of shrimp, both the individual weight gain of each animal and the survival rate during the grow-out period must be maximized in order to produce the maximum gross weight of shrimp. Since disease in general, and gill disease in particular, would be expected to exert a negative influence on both survival rate and individual weight gain, it is of the utmost importance to develop the most effective gill disease treatment process in order to reduce or eliminate this negative influence. In addition, since it appears that the filamentous gill disease organisms L. mucor and Cytophaga sp. appear to significantly enhance the ability of other gill disease forms, especially the algae and the protozoans, to flourish, it is of great importance to develop the most effective chemotherapeutic treatment for reduction of these filamentous organisms.
Thus, while the individual use of either Cutrine-Plus or potassium permanganate is well known to be effective in the treatment of L. mucor and Cytophaga sp. in shrimp, the development of a treatment which shows increased effectiveness is of great importance to the Controlled Environment Aquaculture of shrimp.